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| Home > Publications database > Biochemical and biophysical studies on guanylate cyclase activating protein 1, aCa$^{2+}$-sensor in phototransduction |
| Home > Publications database > Biochemical and biophysical studies on guanylate cyclase activating protein 1, aCa$^{2+}$-sensor in phototransduction |
| Dissertation / PhD Thesis/Book | PreJuSER-24990 |
2001
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/19987
Report No.: Juel-3891
Abstract: The guanylate cyclase activating protein 1 (GCAP-1) is a hydrophobic 24 kDa, Ca$^{2+}$-binding protein with three putative functional EF-hands. Native GCAP-1 contains myristate on the N-terminus $\textit{in vivo}$ and four cysteines. In order to evaluate whether the cysteines in GCAP-1 play a role in its fonction and can be used for monitoring conformational changes, several cysteine-exchange-mutants were constructed. Recombinant wild-type GCAP-1 and the cysteine mutants were successfully overexpressed in $\textit{E. coli}$ and purified by gel filtration and anion exchange chromatography. The single cysteine mutants of GCAP-1 activated ROS-GC1 in a similar fashion as nonmyristoylated wild-type GCAP-1 (half maximal activity, IC$_{50}$, = 5.5 $\mu$M and Hill coefficient, n, = 0.9). The triple cysteine mutants of GCAP-1 activated ROS-GC1 in a similar fashion as myristoylated wild-type GCAP-1 (IC$_{50}$ = 706 riM and n = 1.6). The quad cysteine mutant of GCAP-1 stimulated ROS-GC1 most similarly to native GCAP-1 (IC$_{50}$ = 293 nM and n = 1.7) and also showed an inhibitory effect on ROS-GC1 in high [Ca$^{2+}]_{free}$. Several Ca$^{2+}$-dependent properties of GCAP-1 were examined. On SDS-PAGE, complexing Ca$^{2+}$ with 2 mM EGTA in GCAP-1 solution caused a Cal+-dependent mobility shift of GCAP-1 from approx. 21 kDa to approx. 26 kDa. The $\alpha$-helical content in the secondary structure of GCAP-1 decreased when the nonmyristoylated form was Ca$^{2+}$-free and when the myristoylated form was saturated with Ca$^{2+}$. Furthermore, GCAP-1 existed predominantly as a monomer in the absence of Ca$^{2+}$ and as a dimer in the presence of Ca$^{2+}$. Ca$^{2+}$-bound GCAP-1 was the most thermally stable form, compared with Ca$^{2+}$-free GCAP-1. GCAP-1 showed a pronounced reactivity towards the thiol-modifying reagent, DTNB. When EGTA was added, all four cysteines rapidly reacted with DTNB, while only three cysteines in GCAP-1 were exposed in the presence of CaCl$_{2}$. Cysteine mutants of GCAP-1 were used to investigate the thiol reactivity of every single cysteine in more detail. The cysteines at position 18 and 125 were rapidly exposed in the presence of Ce, whereas the cysteine at position 29 slowly reacted with DTNB in the presence of Ca$^{2+}$. Only the cysteine at position 106 was buried within GCAP-1 when Ca$^{2+}$ was bound, and it was exposed after EGTA addition. Cysteine mutants were also used to study the interaction of GCAP-1 with target regions in ROS-GC1 (i.e. peptides representing hypothetical binding regions). ROS-GCI peptides induced a shielding effect on two cysteines of GCAP-1, when they interacted with GCAP-1.
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